The experiments in this proposal are aimed at investigating the mechanisms whereby metazoan splice sites and exons are constitutively and alternatively recognized during pre-mRNA splicing. In addition to splice sites, exon/intron length and sequence are important parameters for exon recognition. This proposal studies six weak exons or introns that require special sequences to facilitate or regulate their recognition. Three of the studied elements are exonic, three are intronic. Experiments are described to identify and characterize the factors that participate in recognition of these elements during early assembly of the spliceosome. The elements under the study include: Multi-partite exon enhancers from the cardiac troponin T and caldesmon genes that can enhance inclusion of large internal exons and regulate usage of flanking 5' splice sites. Multi-partite exon silencer from HIV that depresses usage of upstream 3' splice sites. Duplicated simple-sequence intron enhancer from cardiac troponin T that compensates for the small size of an adjacent constitutive exon. A G-rich intron enhancer from alpha globin that facilitates splicing and regulates usage of 5' splice sites. A C-rich intron sequence from the small first intron of the Drosophila maleless gene required for recognition of the 5' and 3' splice sites.